Ulva polysaccharide lyase PcPL40A and application thereof

Abstract

The invention relates to ulva polysaccharide lyase PcPL40A and application thereof, and belongs to the technical field of functional enzymes, the amino acid sequence of the ulva polysaccharide lyase PcPL40A is as shown in SEQ ID NO.1, and the coding gene of the ulva polysaccharide lyase PcPL40A is as shown in SEQ ID NO.2 or SEQ ID NO.3. The invention further discloses a preparation method of the ulva polysaccharide lyase PcPL40A. The ulva polysaccharide lyase PcPL40A disclosed by the invention can be used for hydrolyzing ulva to obtain unsaturated ulva disaccharide, tetrasaccharide, hexasaccharide and / or octasaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of functional enzymes, and particularly relates to an ulva polysaccharide lyase PcPL40A and an application thereof. Background Art

[0002] Ulva is a natural macromolecular linear polysaccharide extracted from the cell wall of green algae of the genus Ulva. It is one of the few natural polysaccharides with high sulfate substitution in nature. The main monosaccharide monomers in the structure of Ulva are iduronic acid (IdoA), 3-sulfated rhamnose (Rha3S), xylose (Xyl), 2-sulfated xylose (Xyl2S) and glucuronic acid (GlcA). There are usually four disaccharide units in the structure of Ulva: [→4) β -d-Xyl(1→4)- α -L-Rha3S(1→],[→4) α -L-IdoA(1→4)-α-L-Rha3S(1→],[→4) β -d-GlcA(1→4)- α -L-Rha3S(1→] and [→4) β -d-Xyl2S(1→4)- α -L-Rha3S(1→]. Due to the presence of sulfate groups, Ulva has multiple physiological activities such as antiviral, anti-inflammatory, anticoagulant, and antioxidant. Oligosaccharides obtained by degradation of Ulva have significantly improved solubility, stability, and various physiological activities compared with Ulva polysaccharides, which is an important direction for the high-value utilization of Ulva.

[0003] At present, there are physical, chemical and biological methods for preparing Ulva oligosaccharides. Acid hydrolysis has the advantages of rapid reaction and high substrate concentration, but the products after acid hydrolysis are more complex and difficult to separate. Compared with acid hydrolysis, enzymatic hydrolysis has the advantages of mild reaction, single product and easy separation. Therefore, enzymatic method is a green method with sustainable application prospects. It is of great significance to explore Ulva polysaccharide lyase to prepare Ulva oligosaccharides with specific structures. Ulva polysaccharide lyase degrades Ulva to produce unsaturated oligosaccharides, and unsaturated disaccharides are usually expressed as ∆-Rha3S. The Ulva polysaccharide lyases characterized so far are distributed in the PL24, PL25 and PL28 families, and the main products of the Ulva polysaccharide lyases characterized so far are all tetrasaccharides or disaccharides. There is a serious lack of research on Ulva polysaccharide lyases with larger products, which limits the types of oligosaccharides that can be prepared. Therefore, further expression and characterization of unknown Ulva polysaccharide lyases and development of their potential for the preparation of Ulva polysaccharide lyases with different polymerization degrees are of great significance for in-depth exploration of the structure-activity relationship of oligosaccharides. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a ulva polysaccharide lyase PcPL40A that can degrade ulva, and its main hydrolysis product is unsaturated ulva hexasaccharide. Unsaturated ulva hexasaccharide can be efficiently prepared, which is of great significance for solving the preparation problem of medium- and long-polymerization degree ulva oligosaccharides and has broad application prospects.

[0005] The present invention is achieved through the following technical solutions: One of the purposes of the present invention is to provide a ulva polysaccharide lyase PcPL40A, the amino acid sequence of the ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.1.

[0006] The second object of the present invention is to provide an application of Ulva polysaccharide lyase PcPL40A, wherein the application is to use the Ulva polysaccharide lyase PcPL40A to lyse Ulva to obtain unsaturated Ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides, and the amino acid sequence of the Ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.1.

[0007] The third object of the present invention is to provide an application of the Ulva polysaccharide lyase PcPL40A gene, characterized in that the application is to use the Ulva polysaccharide lyase PcPL40A gene to prepare an enzyme capable of lysing Ulva to obtain unsaturated Ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides, the Ulva polysaccharide lyase PcPL40A gene is shown as SEQ ID NO.2 or SEQID NO.3, and the encoded protein of the Ulva polysaccharide lyase PcPL40A gene is shown as SEQ ID NO.1.

[0008] As a preferred embodiment, the conditions for cracking Ulva are: the enzymatic hydrolysis conditions of Ulva polysaccharide lyase PcPL40A are: substrate concentration is 1-4 g / L, enzyme addition amount is 0.02-0.16 U / mL (1 U represents the amount of enzyme required to release 1 μmol of reducing sugar in 1 minute), preferably 0.08 U / mL, enzymatic hydrolysis temperature is 20-45°C, preferably 40°C, pH value is 6.0-10.0, preferably 8.0, enzymatic hydrolysis time is more than 10 minutes, preferably 12 hours.

[0009] A fourth object of the present invention is to provide an enzyme preparation, which contains ulva polysaccharide lyase PcPL40A, and the amino acid sequence of the ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.1.

[0010] A fifth object of the present invention is to provide an application of the enzyme preparation in hydrolyzing ulva, wherein the application is to use the enzyme preparation to prepare unsaturated ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides.

[0011] Amino acid sequence of Ulva polysaccharide lyase PcPL40A (SEQ ID NO: 1):

[0012] The nucleotide sequence of the gene encoding the Ulva polysaccharidase PcPL40A (direction 5'-3') (SEQ ID NO: 2):

[0013] The beneficial effects of the present invention compared with the prior art: The present invention has found through experimental research that ulva polysaccharide lyase PcPL40A can degrade ulva under the conditions of 40°C and pH 8.0, and the main product composition is unsaturated ulva hexaose, and unsaturated ulva hexaose can be specifically prepared. The present invention constructs an expression vector containing the ulva polysaccharide lyase PcPL40A gene, realizes heterologous expression in Escherichia coli, and provides a good foundation for the industrial large-scale production of the enzyme. The optimal reaction temperature of ulva polysaccharide lyase PcPL40A is 40°C. At this temperature, ulva as a substrate can remain in a solution state, which can better realize the large-scale application of the ulva polysaccharide lyase. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the SDS-PAGE electrophoresis diagram of the purified Ulva polysaccharide lyase PcPL40A of the present invention. Lane M: Protein Maker, Lane P: Pure enzyme Figure 2 It is a line graph showing the effect of temperature change on the hydrolase activity of Ulva polysaccharide lyase PcPL40A; Figure 3 It is a line graph showing the effect of pH change on the hydrolase activity of Ulva polysaccharide lyase PcPL40A; Figure 4 It is a line graph of the remaining enzyme activity after incubation at different temperatures for 1 hour; Figure 5 The curves of residual enzyme activity changes after incubation at 4, 20 and 25 °C for 72 hours; Figure 6 The HPLC chart of the ulva hydrolyzed product by 0.02 U / mL ulva polysaccharide lyase PcPL40A; Figure 7 The HPLC chart of the ulva hydrolyzed product by 0.16 U / mL ulva polysaccharide lyase PcPL40A; Figure 8 The HPLC chart of the separation of the products of Ulva hydrolysis by Ulva polysaccharide lyase PcPL40A; Fig. 9 This is the mass spectrum of the Ulva separation product T1 hydrolyzed by Ulva polysaccharide lyase PcPL40A; Fig.10 This is the mass spectrum of T2, a product of Ulva separated by Ulva polysaccharide lyase PcPL40A; Fig.11 This is the mass spectrum of T3, a product of Ulva separated by hydrolysis of Ulva polysaccharide by Ulva polysaccharide lyase PcPL40A; Fig.12 This is the mass spectrum of T4, a product separated from Ulva hydrolyzed by Ulva polysaccharide lyase PcPL40A; Fig.13 This is the mass spectrum of T5, the product separated from Ulva hydrolyzed by Ulva polysaccharide lyase PcPL40A. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0016] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art. The various terms and phrases used in the present invention have the general meanings known to those skilled in the art.

[0017] The method of the present invention is further described below by means of specific examples.

[0018] Example 1 Cloning of Ulva polysaccharide lyase PcPL40A The marine bacteria mined from the NCBI database in this application Pseudotamlana carrageenivorans The potential Ulva polysaccharide lyase fragment (WP_102996275.1) from the source may have Ulva polysaccharide lyase activity according to the evolutionary tree and multiple sequence comparison analysis. The gene fragment has no homology with the disclosed Ulva polysaccharide lyase, which illustrates the novelty of the sequence mined by the present invention. The gene contains 3483 bases, the sequence is shown in SEQ ID NO.2, and the encoded protein has 1160 amino acids, the sequence is shown in SEQ ID NO.1. According to the codon preference of the host Escherichia coli, the DNA sequence of the gene except the 60bp encoding signal peptide fragment at the 5' end was codon optimized, and the optimized gene sequence is shown in SEQ ID NO.3.

[0019] The nucleotide sequence of the synthetic Ulva polysaccharide lyase PcPL40A encoding gene is as follows (direction 5'-3') (as shown in SEQ ID NO.3): The gene fragment shown in SEQ ID NO.3 was artificially synthesized. PCR amplification was performed using the artificially synthesized gene fragment as a template. The specific primers used in PCR are as follows: Forward primer: 5'-CTGCAACATCCGGTTATTTG -3', as shown in SEQ ID NO.4.

[0020] Reverse primer: 5'-CCCAGAAACTGATTCTGAAA -3', as shown in SEQ ID NO.5.

[0021] Example 2 Construction of an expression vector carrying Ulva polysaccharide lyase PcPL40A The target fragment obtained by amplification in Example 1 was reacted with the linearized pCold Ⅱ vector using a seamless splicing kit at 50 ℃ for 5 minutes, and then transformed into E. coli DH5α competent cells and coated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37 ℃ incubator, a single clone was picked for positive clone verification, and the single clone with the correct band size was sent to a sequencing company for sequencing. The sequencing alignment was completely correct, and the recombinant plasmid was obtained, named pCold- PcPL40A , stored in a -20 ℃ refrigerator for future use.

[0022] Example 3 Construction of an engineered bacterium containing ulva polysaccharide lyase PcPL40A The recombinant plasmid carrying the Ulva polysaccharide lyase gene obtained in Example 2 was transformed into the competent E. coli BL21 (DE3), and the transformation was carried out by 42°C heat shock transformation method, and coated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37°C incubator, a single clone was picked for positive clone verification, and a single clone with the correct band size was cultured overnight in liquid LB medium (containing 100 μg / mL ampicillin). The bacterial solution was stored in 10% glycerol and stored at -80°C for long-term use.

[0023] Example 4 Preparation and purification of Ulva polysaccharide lyase PcPL40A The bacterial liquid preserved in Example 3 was activated by culturing overnight at 37°C in LB liquid culture medium (containing 100 μg / mL ampicillin), then transferred to a 100 mL LB conical flask (containing 100 μg / mL ampicillin), cultured at 37°C, 200 rpm until OD600 was about 0.6, IPTG was added at a final concentration of 0.1 mM, and the culture was transferred to 18°C ​​for 16 hours to express the Ulva polysaccharide lyase PcPL40A.

[0024] After the fermentation is completed, the cells are collected by centrifugation at 8000 rpm for 5 minutes, a certain amount of sterile water is added to wash the cells, and the cells are collected by centrifugation at 8000 rpm for 5 minutes. The cells are re-dissolved in Tris-HCl buffer at pH 8.0 and ultrasonically disrupted under ice bath conditions (320 W, on for 3 seconds, stop for 3 seconds and continue to disrupt for 30 minutes). After complete disruption, centrifuge at 8000 rpm at 4 °C for 15 minutes to collect the supernatant as the crude enzyme. The expressed target gene contains a His purification tag, so we used Ni-NTA affinity chromatography for purification. Different concentration gradients (10, 20, 50, 80, 120, 200 and 500 mM) of imidazole were used to elute the target protein, and the Akta purifier was connected for purification. The SDS-PAGE results showed that a relatively single target protein band could be obtained after gradient concentrations of imidazole ( Figure 1 ). The protein concentration of the purified enzyme solution was determined to be 0.02 g / L by the R250 Coomassie Brilliant Blue method. The enzyme solution was concentrated to a protein concentration of 1.0 g / L using a 50 kDa ultrafiltration tube and ultrapure water as the displacement solvent to obtain pure enzyme for the determination of enzymatic properties.

[0025] Example 5 Determination of the optimal reaction conditions for Ulva polysaccharide lyase PcPL40A The pure PcPL40A enzyme obtained in Example 4 was subjected to different temperatures (20, 30, 40, 45, 50, 60 and 70 °C) to determine the effect of temperature on its hydrolytic enzyme activity. The reaction substrate was 4 g / L Ulva, the pH value was 7.0, and the reaction was terminated by boiling for 10 minutes after 30 minutes of reaction. The reducing sugar released by the reaction was determined by the pHBAH method, that is, 100 μL of the reaction solution was added with 300 μL of pHBAH to terminate the reaction, and then boiled for 5 minutes for color development. After cooling to room temperature, 200 μL was taken to measure the absorbance at 405 nm, and the amount of reducing sugar produced was calculated according to the standard curve (here, the standard curve was drawn with D-galactose as the standard substance). According to the measurement results, it can be seen that PcPL40A shows the greatest hydrolytic activity at 40 °C ( Figure 2 ). The optimal pH of PcPL40A was further determined under the conditions of pH 3-10. Figure 3 As shown, the optimal reaction pH is 8.0.

[0026] Example 6 Determination of the specific enzyme activity of Ulva polysaccharide lyase PcPL40A The enzyme activity determination method is as follows: the reaction system is 200 μL, including pH 8.0, 50 mM Tris-HCl buffer, 4 g / L Ulva, and 1.0 mg pure PcPL40A (prepared in Example 4). The reaction is carried out at 40°C for 10 minutes and then boiled for 10 minutes to terminate the reaction. The reducing sugar released by the reaction is determined by the pHBAH method (see Example 5).

[0027] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of reducing sugar in 1 minute under the optimal reaction conditions.

[0028] It was determined that the specific enzyme activity of Ulva polysaccharide lyase PcPL40A in hydrolyzing Ulva was 0.31 U / mg.

[0029] Example 7 Determination of thermal stability of Ulva polysaccharide lyase PcPL40A The pure PcPL40A enzyme obtained in Example 4 was incubated at 20, 30, 40, 50, 60, 70 and 80°C for 60 minutes and then its residual enzyme activity was measured. Figure 4 As shown, the results showed that PcPL40A still maintained a relative enzyme activity of about 60.58% after incubation at 30 °C for 60 min.

[0030] The pure PcPL40A enzyme was further incubated at 4, 20, and 25 °C for 72 hours, and samples were taken over time to determine the residual enzyme activity. Figure 5 As shown, surface PcPL40A still retained 47.54% and 43.39% of the initial enzyme activity after incubation at 4 ℃ and 25 ℃ for 72 hours, respectively.

[0031] Example 8 Determination of the hydrolysis products of Ulva hydrolyzed by PcPL40A The pure PcPL40A enzyme obtained in Example 4 was reacted with 4 g / L of Ulva at 40°C and pH 8.0 for 12 hours, and the hydrolysis product was determined by HPLC. Figure 6 As shown in the figure, no oligosaccharide products were generated at 0 minutes. As the reaction proceeded, peaks P2 and P3 were generated first and gradually accumulated, and finally five oligosaccharide peaks P1, P2, P3, P4 and P5 were generated, of which P2 and P3 were the main products. Figure 7 As shown, no oligosaccharide products were generated at 0 minutes, and six oligosaccharide peaks of P1, P2, P3, P4, P5 and P6 were finally produced, among which P2 and P3 were still the main products.

[0032] Due to the lack of Ulva oligosaccharide standards on the market, it is impossible to determine what oligosaccharides the six oligosaccharide peaks P1, P2, P3, P4, P5 and P6 are. We used gel chromatography to separate them, such as Figure 8 As shown in the figure, a total of five oligosaccharides were obtained, T1 corresponds to P1, T2 contains both P2 and P3, T3 corresponds to P4, T4 corresponds to P5, and T5 corresponds to P6. T1, T2, T3, T4 and T5 were further detected by mass spectrometry: Fig. 9 As shown, T1 is an unsaturated octasaccharide; Fig.10 As shown, T2 is an unsaturated hexasaccharide; Fig.11 and Fig.12 As shown, T3 and T4 are both unsaturated tetrasaccharides; Fig.13 As shown, T5 is an unsaturated disaccharide. This indicates that PcPL40A cleaves Ulva to produce unsaturated disaccharides, tetrasaccharides, hexasaccharides and octasaccharides, among which unsaturated hexasaccharides are the main products.

Claims

1. A Ulva polysaccharide lyase PcPL40A, characterized in that: The amino acid sequence of the Ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.

1.

2. An application of Ulva polysaccharide lyase PcPL40A, characterized in that: The application is to use the ulva polysaccharide lyase PcPL40A to lyse ulva to obtain unsaturated ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides. The amino acid sequence of the ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.

1.

3. An application of a Ulva polysaccharide lyase PcPL40A gene, characterized in that: The application is to use the Ulva polysaccharide lyase PcPL40A gene to prepare an enzyme capable of lysing Ulva to obtain unsaturated Ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides, the Ulva polysaccharide lyase PcPL40A gene is shown as SEQ ID NO.2 or SEQ ID NO.3, and the encoded protein of the Ulva polysaccharide lyase PcPL40A gene is shown as SEQ ID NO.

1.

4. The use according to claim 2 or 3, characterized in that: The conditions for cracking Ulva are as follows: the enzymatic hydrolysis conditions of Ulva polysaccharide lyase PcPL40A are as follows: substrate concentration is 1-4 g / L, enzyme addition amount is 0.02-0.16 U / mL, enzymatic hydrolysis temperature is 20-45°C, pH value is 6.0-10.0, and enzymatic hydrolysis time is more than 10 minutes.

5. The use according to claim 4, characterized in that: The substrate concentration was 1-4 g / L, the enzyme addition amount was 0.08 U / mL, the enzymatic hydrolysis temperature was 40 °C, the pH value was 8.0, and the enzymatic hydrolysis time was 12 hours.

6. An enzyme preparation, characterized in that The enzyme preparation contains ulva polysaccharide lyase PcPL40A, and the amino acid sequence of the ulva polysaccharide lyase PcPL40A is shown in SEQ ID NO.

1.

7. Use of the enzyme preparation according to claim 6 in hydrolyzing Ulva, characterized in that: The application is to use the enzyme preparation to prepare unsaturated ulva disaccharide, tetrasaccharide, hexasaccharide and / or octasaccharide.

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